A number of closely related species of parasitic copepods in the family Caligidae (caligid copepods) infect and cause disease in cultured fish. Collectively, these species are referred to as sea lice. There are three major genera of sea lice: Pseudocaligus, Caligus and Lepeophtheirus. With respect to salmonid production throughout the northern hemisphere, one species, the salmon louse (Lepeophtheirus salmonis), is responsible for most disease outbreaks on farmed salmonids. This parasite is responsible for indirect and direct losses in aquaculture in excess of US $100 million annually (Johnson, S. C., et al., Zool Studies 43: 8-19, 2004). All developmental stages of sea lice, which are attached to the host, feed on host mucus, skin and blood. The attachment and feeding activities of sea lice result in lesions that vary in their nature and severity depending upon: the species of sea lice, their abundance, the developmental stages present and the species of the host (Johnson, S. C. et al., “Interactions between sea lice and their hosts”. In: Host-Parasite Interactions. Editors: G. Wiegertjes and G. Flik, Garland Science/Bios Science Publications, 2004, pp. 131-160). In the southern hemisphere, Caligus rogercresseyi, is the primary caligid affecting the salmon farming industry in Chile (González, L. and Carvajal, J. Aquaculture 220: 101-117, 2003).
Caligid copepods have direct life cycles consisting of two free-living planktonic nauplius stages, one free-swimming infectious copepodid stage, four to six attached chalimus stages, one or two preadult stages, and one adult stage (Kabata, Z., Book 1: Crustacea as enemies of fishes. In: Diseases of Fishes., Editors: Snieszko, S. F. and Axelrod, H. R.; New York, T.F.H. Publications, 1970, p. 171). Each of these developmental stages is separated by a moult. Once the adult stage is reached caligid copepods do not undergo additional moults. In the case of L. salmonis, eggs hatch into the free-swimming first nauplius stage, which is followed by a second nauplius stage, and then the infectious copepodid stage. Once the copepodid locates a suitable host fish it continues its development through four chalimus stages, first and second preadult stages, and then a final adult stage (Schram, T. A. “Supplemental descriptions of the developmental stages of Lepeophtheirus salmonis (Krøyer, 1837) (Copepoda: Caligidae)”. In: Pathogens of Wild and Farmed Fish: Sea Lice. Editors: Boxshall, G. A. and Defaye, D., 1993, pp. 30-50). The moults are characterized by gradual changes as the animal grows and undertakes physical modifications that enable it to live as a free-roaming parasite, feeding and breeding on the surface of the fish.
Caligid copepods (sea lice) feed on the mucus, skin and blood of their hosts leading to lesions that vary in severity based on the developmental stage(s) of the copepods present, the number of copepods present, their site(s) of attachment and the species of host. In situations of severe disease, such as is seen in Atlantic salmon (Salmo salar) when infected by high numbers of L. salmonis, extensive areas of skin erosion and hemorrhaging on the head and back, and a distinct area of erosion and sub-epidermal hemorrhage in the perianal region can be seen (Grimnes, A. et al. J Fish Biol 48: 1179-1194, 1996). Sea lice can cause physiological changes in their hosts including the development of a stress response, reduced immune function, osmoregulatory failure and death if untreated (Johnson et al., supra).
There are several management strategies that have been used for reducing the intensity of caligid copepod (sea lice) infestations. These include: fallowing of sites prior to restocking, year class separation and selection of farm sites to avoid areas where there are high densities of wild hosts or other environmental conditions suitable for sea lice establishment (Pike, A. W. et al. Adv Parasitol 44: 233-337, 1999). Although the use of these strategies can in some cases lessen sea lice infection rates, their use individually or in combination has not been effective in eliminating infection.
A variety of chemicals and drugs have been used to control sea lice. These chemicals were designed for the control of terrestrial pests and parasites of plants and domestic animals. They include compounds such as hydrogen peroxide, organophosphates (e.g., dichlorvos and azamethiphos), ivermectin (and related compounds such as emamectin benzoate), insect molting regulators and pyrethrins (MacKinnon, B. M., World Aquaculture 28: 5-10, 1997; Stone J., et al., J Fish Dis 22: 261-270, 1999). Sea lice treatments can be classified into those that are administered by bath (e.g. organophosphates, pyrethrins) and those administered orally (e.g. ivermectin). Bath treatments for sea lice control are difficult, expensive to apply and can have significant effects of fish growth following treatments (MacKinnon, supra). Chemicals used in bath treatments are not necessarily effective against all of the stages of sea lice found on fish. At present the use of oral treatments such as SLICE® (emamectin benzoate) is predominant in the salmonid industry. Unlike chemicals administered as bath treatments SLICE® does provide short-term protection against re-infection. This treatment although easier to apply than bath treatments is still expensive and, like bath treatments, requires a withdrawal period before animals can be slaughtered for human consumption (Stone, supra). As seen in terrestrial pest and parasites there is evidence for the development of resistance in L. salmonis to some of these treatments, especially in frequently-treated populations (Denholm, I., Pest Manag Sci 58: 528-536, 2002). To reduce the costs associated with sea lice treatments and to eliminate environmental risks associated with these treatments new methods of sea lice control such as vaccines are needed.
A characteristic feature of attachment and feeding sites of caligid copepods on many of their hosts is a lack of a host immune response (Johnson et al., supra; Jones, M. W., et al., J Fish Dis 13: 303-310, 1990; Jónsdóttir, H., et al., J Fish Dis 15: 521-527, 1992). This lack of an immune response is similar to that reported for other arthropod parasites such as ticks on terrestrial animals. In those instances suppression of the host immune response is due to the production of immunomodulatory substances by the parasite (Wikel, S. K., et al., “Arthropod modulation of host immune responses”. In The Immunology of Host-Ectoparasitic Arthropod Relationships. Editors: Wikel, S. K., CAB Int., 1996, pp. 107-130). These substances are being investigated for use as vaccine antigens to control these parasites. Sea lice, such as L. salmonis, like other arthropod ectoparasites, produce biologically active substances at the site of attachment and feeding that limits the host immune response. As these substances have potential for use in a vaccine against sea lice we have identified a number of these substances from L. salmonis and have examined their effects of host immune function in vitro.
Potential antigens have been identified using a combination of molecular biological, proteomic, biochemical and immunological techniques. For example, an increase in protease activity has been observed in the mucus of L. salmonis infected Atlantic salmon, compared to non-infected fish (Ross, N. W., et al., Dis Aquat Org 41: 43-51, 2000; Fast, M. D., et al., Dis Aquat Org 52: 57-68, 2002). This increased activity is primarily due to the appearance of a series of low molecular weight (18-24 kDa) proteins, that are produced by L. salmonis and were identified as trypsins based on activity, inhibition studies and size. Trypsin activity was identified in infected salmon mucus using aminobenzamidine affinity adsorption and protease zymography (Firth, K. J., et al., J Parasitol 86: 1199-1205, 2000). Several genes encoding for trypsin have been characterized from L. salmonis and the site of trypsin expression determined (Johnson, S. C., et al., Parasitol Res 88: 789-796, 2002; Kvamme, B. O., et al., Int. J. Parasitol. 34, 823-832, 2004; Kvamme, B. O. et al., Gene 352:63-72, 2005).
Several cDNA libraries have been developed from the copepodid, preadult and adult stages of L. salmonis. An expressed sequence tag (EST) study of the preadult library resulted in the identification of a number of genes encoding trypsin and related proteases (including chymotrypsin and others in the peptidase S1 family), heat shock proteins, cuticle proteins and metabolic enzymes. Some of these genes as described herein have utility as antigens in a sea lice vaccine.
Trypsin-like activity is secreted by L. salmonis onto the salmon skin and is believed to be used by the sea lice to feed on the salmon mucus, skin and blood and to protect the sea lice from the salmon immune response (Firth, et al. supra). Trypsin was discovered in the secretion products (SPs) of sea lice, following stimulation with dopamine, by amino acid sequencing using mass-spectrometry. Table 1 shows the peptide sequences of L. salmonis secreted trypsin. Protection against sea lice trypsin may reduce the feeding of the lice and reduce the suppression of the immune response.
TABLE 1Summary of L. salmonis secreted trypsin identified from LC/MS/MSAssoc.Sea LiceFractionParentprotein(pool#-IonErrorPeptide sequencematchesfraction#)(m/z)Mr (Da)(ppm)aScoreb(Start-end)cSea Lice1-2579.801157.772746215FIDWIAEHQ223Trypsin(SEQ ID NO: 25)(types 1-1-1638.351274.69387271IAVSDITYHEK814)(SEQ ID NO: 26)3-6920.181840.281325115DQEFIGDVVVSGWGTISSSGPPSPVLK141(SEQ ID NO: 27) SL-09031-1580.281158.484627NQYDEFESKvitellogenin-(SEQ ID NO: 28)likeSL-14691-1724.851447.661724LSFEHETTEEARSEP protein 3(SEQ ID NO: 29)1-2879.981757.912972IILGHEFTPGYIENR(SEQ ID NO: 30) SL-05471-1604.311204.671925IVILKELSSGMSEP protein 1(SEQ ID NO: 31) +M oxidationd SL-08581-21248.712495.336535AGQYGGEISGIVLPNIPSEP Protein 2PSISNLAK(SEQ ID NO: 32)aDifference (in parts-per-million) between measured mass and mass predicted from the DNA sequence.bScore from MASCOT ™ search, scores above 21 indicate identity or extensive homology (p < 0.05)cCyanogen bromide/tryptic peptide sequence predicted from the DNA sequence.d+M oxidation means that the MASCOT match was for a peptide containing an oxidized methionine residue.
Vitellogenin-like protein was discovered in the secretion products (SPs) of sea lice following stimulation with dopamine. Vitellogenin has previously been reported as an effective antigen in a tick vaccine (Tellam, R. I., et al., Vet Parasitol 103: 141-156, 2002). Inclusion of vitellogenin in a sea lice vaccine may interfere with the fecundity of sea lice and reduce the number of offspring and hence reduce future numbers of sea lice. In addition, vitellogenin-like proteins have been implicated in the synthesis of melanin in invertebrates (Lee, K. M. et al., Eur J Biochem 267:3695-3703, 2000). Melanin is an important defence molecule of invertebrates.
Mussel adhesion-like genes express proteins similar to those found in the mussel byssus threads that mussels use to attach themselves to solid surfaces. How these genes relate to sea lice infestation is not currently understood, but they may be involved in the production of frontal filaments. The frontal filament is used by chalimus stages to physically attach themselves to the host (Gonzalez-Alanis, P., et al., J Parasitol 87: 561-574, 2001).
BCS-1 genes are expressed by barnacles when they switch from a planktonic form to an attached form (Okazaki, Y., et al., Gene 250 (1-2): 127-135, 2000). There is currently evidence to suggest that these are cuticle-binding proteins. Disruption of these proteins by antibodies may interfere with moulting, integrity of the sea lice cuticle and normal growth of the lice.
Secretory proteins produced by the sea lice may act as immunomodulatory agents or assist in the feeding activities on the host (Fast, M. D., et al., Exp Parasitol. 107:5-13, 2004; Fast, M. D., et al., J Parasitol 89: 7-13, 2003). Neutralization of these activities by host-derived antibodies may impair sea lice growth and survival on salmon.
Vaccines are generally safer than chemical treatments, both to the fish and to the environment. However, no commercial vaccines against sea lice have been developed to date. Vaccine development has been hindered by a lack of knowledge of the host-pathogen interactions between sea lice and their hosts. There appears to be very limited antibody response in naturally infected hosts. Experimental vaccines, particularly through whole-animal extracts, have been produced against L. salmonis. Investigations in the development of sea lice vaccines have targeted immunogenic proteins from sea lice and, in particular, targeting gut antigens. These vaccines, based on whole animal extracts, have not been shown to be protective though their administration did result in minor changes in L. salmonis fecundity (Grayson T. H., et al., J Fish Biol 47: 85-94, 1995). This particular study, however, was a one-time trial and no further results have been reported from this group. Liposome-based fish vaccines in certain species of fin-fish have also been explored (Keough, PCT Application WO 03/101482) but not in combination with sea lice antigens.
A more recent discussion of possible vaccine targets in the gut was put forth by Raynard et al.; however, their studies have been met with limited success (Raynard, R. S., et al., Pest Manag Sci 58: 569-575, 2002).
Promiscuous T-Cell Epitopes
Promiscuous T-cell epitopes (or “PTC epitopes”) are highly immunogenic peptides that can be characterized in part by their capacity to bind several isotypic and allotypic forms of human MHC class II molecules. By helping to bypass MHC restriction, they can induce T-cell and antibody responses in members of a genetically diverse population expressing diverse MHC haplotypes. The PTC epitopes can therefore be combined with antigens that, by themselves, are poorly immunogenic, to generate potent peptide immunogens. In the present invention, these epitopes are incorporated into the composition to enhance the immunogenicity of the antigen, and the composition overall, in a broad range of species.
Promiscuous T-cell epitopes can be derived from naturally occurring immunogens of viral and bacterial origin. Naturally occurring PTC epitopes can also be conservatively modified by single- or multiple-amino acid additions, deletions or substitutions (e.g. within classes of charged, hydrophilic/hydrophobic, steric amino acids) to obtain candidate sequences that can be screened for their ability to enhance immunogenicity.
Non-naturally occurring PTC epitopes can be artificially synthesized to obtain sequences that have comparable or better immunogenicity. Artificial PTC epitopes can range in size from about 15 to about 50 amino acid residues in length and can have structural features such as amphipathic helices, which are alpha-helical structures with hydrophobic amino acid residues dominating one face of the helix and charged or polar residues dominating the surrounding faces. The PTC epitopes may also contain additional primary amino acid patterns, such as a Gly or a charged residue followed by two to three hydrophobic residues, followed in turn by a charged or polar residue (a Rothbard sequence). In addition, PTC epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth, and eighth positions after the charged residue.
These features may be incorporated into the designs of artificial PTC epitopes. Variable positions and preferred amino acids are available for MHC-binding motifs (Meister et al., Vaccine, 1995; 13:581-591). For example, the degenerate PTC epitope described in WO 95/11998 as SSAL1TH1 has the degenerate sequence (Asp/Glu)-(Leu/Ile/Val/Phe)-Ser-(Asp/Gly)-(Leu/Ile/Val/Phe)-(Lys/Arg)-Gly-(Leu/Ile/Val/Phe)-(Leu/Ile/Val/Phe)-(Leu/Ile/Val/Phe)-His-(Lys/Arg)-Leu/Ile/Val/Phe)-(Asp/Glu)-Gly-(Leu/Ile/Val/Phe)-.
Specific Examples of PTC Epitopes
Particularly useful promiscuous T-cell epitopes are measles virus protein F LSEIKGVIVHRLEGV (SEQ ID NO: 33); or tetanus sequence QYIKANSKFIGITEL (SEQ ID NO: 34).
Examples of particularly useful promiscuous T-cell epitopes are listed in Table 2:
TABLE 2Examples of Promiscuous T-cell EpitopesSEQ IDdescriptionamino acid sequenceNO:measles 289-302LSEIKGVIVHRLEGV33 tetanus toxin 830-844QYIKANSKFIGITEL34
Because of a lack of understanding of the mechanisms and pathology surrounding sea lice infestation of salmon, identification of suitable targets to treat the disease has not been successful. This has hampered the progress of vaccine research and as such, despite the promise and success of vaccine-based therapies in other areas of infection, a suitable sea lice vaccine has yet to be developed. Consequently, there is a need to provide effective suitable molecular targets (antigens) and a vaccine against sea lice infection.